A detailed study of the subunit association of human hemoglobin is being undertaken. The aims are to gain a better understanding, by measuring the dimer-tetramer association behavior of the molecule, of the cooperativity and interligand linkage displayed by the molecule. These measurements will be carried out at different pressures or concentrations of the heme ligand. The role of subunit association in mediating the reciprocal (linked) affinity of heme ligands and non-heme ligands will also be explored. Within this context, the conformational requirements for gelation of hemoglobin S will be further examined and the role of divalent cations in the gelation process will be explored. This work will exploit the availability of hemoglobins which have been specifically carbamylated with cyanate at either their alpha- or beta-chain amino terminal groups in order to gain chain-specific information about the sites of action of the non-heme ligands. The primary method to be employed is equilibrium ultracentrifugation. A recently built computer-controlled absorption scanner for the ultracentrifuge will provide the sensitivity and precision necessary to the experiments. For the work at partial heme saturation, novel and flexible methods of data analysis are proposed and new ultracentrifuge cells of inert and oxygen-impermeable materials will be constructed.